Designation D7463 − 16´1 Standard Test Method for Adenosine Triphosphate (ATP) Content of Microorganisms in Fuel, Fuel/Water Mixtures, and Fuel Associated Water1 This standard is issued under the fixe[.]
Trang 1Designation: D7463−16
Standard Test Method for
Adenosine Triphosphate (ATP) Content of Microorganisms
This standard is issued under the fixed designation D7463; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision A number in parentheses indicates the year of last reapproval A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
ε 1 NOTE—Subsection 12.4 was corrected editorially in October 2016.
1 Scope*
1.1 This test method provides a protocol for capturing,
concentrating, and testing the adenosine triphosphate (ATP)
present in a fuel system sub-sample (that is, test specimen)
associated with:
1.1.1 Microorganisms and hydrophilic particles found in
liquid fuels as described in Table X6.1, or
1.1.2 Microorganisms and hydrophilic particles found in
mixture of fuel and associated bottom water or just associated
bottom water
1.1.3 ATP detected by this bioluminescence test can be
derived from cellular ATP, extra-cellular ATP, or some
combi-nation of both
1.1.4 Cellular and extra-cellular ATP utilized to perform
ATP bioluminescence are captured and concentrated from a
fuel system sample into an aqueous test specimen (that is,
sub-sample) for testing For example, for a fuel system sample
that does not contain any visible fuel associated bottom water,
the aqueous test specimen is the capture solution itself
de-scribed in8.2.1.1 For fuel system samples that are a mixture
of fuel and associated bottom water (that is, free water), the test
specimen is an aliquant of the capture solution and associated
bottom water
1.2 The ATP is measured using a patented bioluminescence
enzyme assay, whereby light is generated in amounts
propor-tional to the concentration of ATP in the sample The light is
produced and measured quantitatively using dedicated ATP test
pens2 and a dedicated luminometer2 and reported in
(instru-ment specific) Relative Light Units
1.3 This test method is equally suitable for use in the laboratory or field
1.4 Although bioluminescence is a reliable and proven technology, this method does not differentiate ATP from bacteria or fungi
1.5 For water or capture solution samples, the concentration range of ATP detectable by this test method is 1 × 10–11M to
3 × 10–8M which is equivalent to 1 × 10–14moles/mL to 3 ×
10–11moles/mL for water samples or capture solution Assum-ing testAssum-ing on fuel phase is performed on a 500 mL volume of fuel the equivalent concentrations is fuel would be: 6 × 10–11
M to 2 × 10–14M
1.6 The values stated in SI units are to be regarded as standard No other units of measurement are included in this standard
1.6.1 There is one exception—Relative Light Unit (RLU) as defined in3.1.19
1.7 This standard does not purport to address all of the
safety concerns, if any, associated with its use It is the responsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.
2 Referenced Documents
2.1 ASTM Standards:3
D396Specification for Fuel Oils
D975Specification for Diesel Fuel Oils
D1655Specification for Aviation Turbine Fuels
D2880Specification for Gas Turbine Fuel Oils
D4012Test Method for Adenosine Triphosphate (ATP) Con-tent of Microorganisms in Water
D4175Terminology Relating to Petroleum Products, Liquid Fuels, and Lubricants
D6300Practice for Determination of Precision and Bias
1 This test method is under the jurisdiction of ASTM Committee D02 on
Petroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility of
Subcommittee D02.14 on Stability and Cleanliness of Liquid Fuels.
Current edition approved June 1, 2016 Published June 2016 Originally
approved in 2008 Last previous edition approved in 2015 as D7463 – 15 DOI:
10.1520/D7463-16E01.
2 The sole source of supply, repair, recertification, and technical support of the
apparatus or test pen known to the committee at this time is Merck KGaA, 64271
Darmstadt, Germany (Worldwide) or Fuel Quality Services, Inc., 4584 Cantrell Rd.,
Flowery Branch, GA 30542 (USA) If you are aware of alternative suppliers, please
provide this information to ASTM International Headquarters Your comments will
receive careful consideration at a meeting of the responsible technical committee, 1
which you may attend.
3 For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
*A Summary of Changes section appears at the end of this standard
Trang 23.1.1 For definition of terms used in this test method, refer
to Terminology D4175
3.1.2 adenosine triphosphate, n—molecule comprised of a
purine and three phosphate groups, that serves as the primary
energy transport molecule in all biological cells
3.1.3 adenosine monophosphate, n—molecule formed by
the removal of two (2) molecules of phosphate (one
pyrophos-phate molecule) from ATP
3.1.4 aseptic, adj—sterile, free from viable microbiological
contamination
3.1.5 bioluminescence, n—production and emission of light
by a living organism as the result of a chemical reaction during
which chemical energy is converted to light energy
3.1.6 biomass, n—biological material including any
mate-rial other than fossil fuels which is or was a living organism or
component or product of a living organism
3.1.7 capture solution, n—aqueous solution of proprietary
composition used to capture and concentrate hydrophilic
com-pounds and particles from liquid fuels
3.1.8 cellular adenosine triphosphate (cellular-ATP),
n—ATP present in whole cells, whether they are living or dead.
3.1.8.1 Discussion—Cellular-ATP is released upon
inten-tional lysis (rupturing) of microbial cells during the sample
preparation process Microbially infected fluids contain both
cellular (cell-associated/cell-bound) and extra-cellular ATP
3.1.9 culturable, adj—microorganisms that proliferate as
indicated by the formation of colonies in or on solid growth
media, or the development of turbidity in liquid growth media
under specified growth conditions
3.1.10 extracellular ATP, n—ATP that is not contained
inside a cell
3.1.10.1 Discussion—ATP is released into the environment
when cells die and break open (lyse), for example, as when
they are killed by exposure to some microbicides ATP released
into the environment can persist for several days after a cell has
been lysed Consequently extracellular ATP must be subtracted
from total ATP to determine the concentration of viable
cell-associated (biomass associated) ATP However,
extracel-lular ATP can also be an indicator of “distant” biomass, for
example, biofilm in the system
3.1.11 free water, n—undissolved water present in a
hydro-phobic material
3.1.11.1 Discussion—Free water in fuel such as
hydrocar-bon diesel fuel can be present as a suspended haze, as droplets
on the walls of the vessel, or as a separate layer on the bottom
of the vessel
3.1.14 invert emulsion layer, n—interface between the water
phase and fuel phase of a fuel water sample which consists of water micelles dispersed in the fuel
3.1.15 luciferase, n—general term for a class of enzymes
that catalyze bioluminescent reactions
3.1.16 luciferin, n—general term for a class of light-emitting
biological pigments found in organisms capable of biolumi-nescence
3.1.17 luminometer, n—instrument capable of measuring
light emitted as a result of non-thermal excitation
3.1.18 pyrogen free, n—free of substances which can induce
fever
3.1.19 relative light unit (RLU), n—instrument-specific unit
of measurement reflecting the number of photons emitted by the Luciferin-Luciferase driven hydrolysis of ATP to AMP plus pyrophosphate
3.1.19.1 Discussion—RLU is not an SI unit, however, RLU
are proportional to ATP concentration
3.1.20 test specimen, n—a representative piece of a sample 3.1.20.1 Discussion—For this test method, the test specimen
is an aqueous sub-sample drawn from the fuel system sample that is tested for the presence of cellular and/or extra-cellular ATP In the case of a fuel system sample that is fuel only in the absence of associated bottom water, the test specimen is the capture solution (3.1.7) For fuel system samples that contain associated bottom water, the test specimen is an aliquant of the capture solution and associated bottom water (3.1.11)
3.1.21 viable microbial biomass, n—metabolically active
(living) micro-organisms
3.2 Abbreviations:
3.2.1 AMP—adenosine monophosphate 3.2.2 ATP—adenosine triphosphate 3.2.3 HDPE—high density polyethylene 3.2.4 NAD + —nicotinamide adenine dinucleotide, oxidized
form
3.2.5 NADH—nicotinamide adenine dinucleotide, reduced
form
3.2.6 NADP + —nicotinamide adenine dinucleotide phosphate, oxidized form
3.2.7 NADPH—nicotinamide adenine dinucleotide phosphate, reduced form
3.2.8 PP—polypropylene 3.2.9 RLU—relative light units
Trang 34 Summary of Test Method
4.1 A fuel system sample is obtained either for condition
monitoring or for diagnostic testing, for example, fuel from a
fuel system that is exhibiting problems such as sediment
formation or filter plugging where the presence of
micro-organisms is suspected
4.2 Microbial ATP is captured from the fuel system sample,
concentrated into a test specimen, and tested using a
biolumi-nescence reaction The light generated by the lumibiolumi-nescence
reaction is proportional to the amount of ATP present in the test
specimen as measured in a luminometer.2
4.3 Test results should be documented for evaluation and
trending
4.4 Specialized test methods for fuel samples, water
samples, extracellular determination, or resolving potential
matrix interference in bottom water samples are described in
Appendix X4 andAppendix X5
5 Significance and Use
5.1 This test method measures the concentration of ATP
present in the sample ATP is a constituent of all living cells
including bacteria and fungi Consequently, the presence of
ATP is a reliable indicator of microbial contamination in fuel
systems ATP is not associated with matter of non-biological
origin
5.2 This test method differs from Test Method D4012 as
follows:
5.2.1 By providing for the rapid determination of ATP
present in a fuel (petroleum) sample, a fuel and water mixture
sample, fuel-associated bottom water sample, and extracellular
ATP freely available in the fuel or aqueous sample matrix;
5.2.2 By providing for a method to capture, extract, and
quantify ATP using self-contained test device and
luminom-eter;
5.2.3 By providing a method of quantifying ATP present in
fuel or water matrices in generally less than 10 min; and
5.2.4 By providing for the rapid separation of the ATP from
chemical interferences that have previously prevented the use
of ATP determinations in complex fluids containing
hydrocar-bons and other organic molecules
5.3 This test method does not require the use of hazardous
materials and does not generate biohazard waste
5.4 This test method can be used to estimate viable micro-bial biomass, to evaluate the efficacy of antimicromicro-bial pesticides, and to monitor microbial contamination in fuel storage and distribution systems
6 Interferences
6.1 Sample containers and sampling devices shall be clean and free of both ATP and microbial contamination
6.2 Ensure that the sampling stick on the ATP Test Pen does not come into contact with any contaminating surfaces Con-tact with a surface or substance can cause contamination with high levels of ATP, giving erroneous results
6.3 Luciferase is an enzyme, which can be inhibited or denatured by high temperatures, the presence of heavy metals, and high salt concentrations in the sample These conditions are unlikely to occur except in samples containing large volumes of bottom-water samples from storage tanks and similar systems
6.3.1 For samples in which inhibition is suspected or likely
to occur, testing of a dilution of the sample is described in
Appendix X4
7 Apparatus
7.1 An example of the luminometer2is shown as a diagram
inFig 1
7.2 Warning—The apparatus is not explosion-proof The
instrument should not be operated in explosive atmospheres or
in locations where there may be explosive fumes, as it cannot
be grounded
7.3 Sample bottle, round wide-mouth, nominal capacity
500 mL or 1000 mL, HDPE (High Density Poly Ethylene) or equivalent There shall be sufficient excess volume in the sample bottle so that there is at least 10 % head space in addition to the 500 mL or 1000 mL sample volume to facilitate the shearing and mixing of the capture solution
7.3.1 Sample bottles may be reused provided they are cleaned and dried correctly Refer to test supplier’s information regarding recommended cleaning procedure
7.4 Pipettors, fixed volume or adjustable, capable of provid-ing discrete volumes of bottom water to determine the presence
of matrix interference as described inAppendix X4 Example pipettor volumes include 10 µL, 50 µL, and 100 µL
FIG 1 Luminometer
Trang 48 Reagents and Materials
8.1 Reagents:
8.1.1 ATP di-sodium salt.
8.1.2 Water, Pyrogen free.
8.2 Materials:
8.2.1 ATP test pens:2
8.2.1.1 HY-LiTE4Fuel Test Pen, as shown in Fig 2
8.2.1.2 HY-LiTE4Free ATP Pen, as shown inFig 2
8.2.2 Pasteur pipettes, sterile, disposable, polyethylene,
1.0 mL
8.2.3 Pasteur pipettes sterile, disposable, polyethylene,
10.0 mL
9 Sampling, Test Specimens, and Test Units
9.1 Samples shall be drawn in accordance with Practice
D7464and dispensed into a clean 500 mL sample bottle (7.3)
9.2 Aircraft fuel systems shall be drawn in accordance with
the applicable Aircraft Maintenance Manual and dispensed into
a clean 1000 mL sample bottle (7.3)
9.3 To reduce the risk of accidental contamination, samples
intended for microbiological testing shall not be used for other
tests until after they are no longer needed for microbiological
testing
9.4 It may be possible to accidentally cross contaminate the
sample under field conditions To reduce risk of potential
cross-contamination, rinse the sample device(s) and sample
container(s) with a 70 % alcohol (isopropyl alcohol or ethanol)
and water solution and let air dry All devices (except factory
new, clean bottles) should be disinfected in this manner to
minimize the likelihood of cross-contamination Use care to
not touch the interior of the freshly decontaminated sample
devices or sample bottles Remove the container lid
immedi-ately before dispensing the sample into the container and
replace the lid on the container as soon as possible
9.5 Microbial contaminant populations are dynamic Mi-crobes within the sample can proliferate or die during the interval between collection and testing Consequently, samples shall be processed within 24 h after collection
9.6 If samples are to be tested later than 4 h after collection, store the samples either on ice or refrigerated at >0 °C to 5°C until tested Avoid freezing samples Allow samples to equili-brate to room temperature before testing
10 Calibration and Standardization
10.1 The luminometer2, which is specific to this test, is factory calibrated and temperature compensated to give a linear response from 0 to 99 000 RLU at temperatures between 5 °C and 35 °C (41 °F and 95 °F) No calibration is necessary because calibrations checks are performed automatically dur-ing start-up
10.2 RLU data may be converted to ATP concentration by interpolating from a standard curve as described in Appendix X5
10.3 1 RLU is equivalent to approximately 5 × 10–15grams ATP
11 Procedure
11.1 Analysis of Fuel and Combined Fuel and Water
Samples:
11.1.1 Collect sample according to9.1or9.2 11.1.1.1 If the sample is Fuel-Only, dispense 500 mL into a clean sample bottle (7.3) for testing
11.1.1.2 If the sample is a Fuel/Water Mixture or Water-Only, dispense 400 mL to 500 mL into a clean sample bottle (7.3) for testing
11.1.1.3 If the sample is aviation fuel, dispense 1000 mL into a clean sample bottle (7.3) for testing
11.1.1.4 If total sample is >500 mL, transfer 500 mL to a clean sample bottle (7.3)
11.1.2 Obtain the small pipette from the fuel test kit
4 Registered trademark of Merck KGaA, 64271 Darmstadt, Germany.
FIG 2 Fuel Test Pen and Free ATP Test Pen
Trang 511.1.3 Using a clean implement (for example, scissors or
knife), cut the protective plastic sleeve open at the bulb-end
and remove the pipette Do not touch the tip and lower stem of
the pipette by hand or against any surfaces
11.1.4 Using the small sterile pipette, transfer the capture
solution from the fuel test pen reservoir into the bottle that
contains the sample
11.1.5 Rinse the interior of the pipette with sample to ensure
maximum transfer of the capture solution to the sample
11.1.6 Dispose of the pipette as solid (fuel-contaminated)
waste according to local regulations
11.1.7 Close the lid securely on the sample vessel
11.1.8 Shake the sample vigorously for 30 s
11.1.9 Place the sample vessel on level surface and let stand
for 5 min
N OTE 1—The capture solution will readily dissolve into the free water
associated with the fuel sample or those samples that contain only water.
The presence of water in the sample will cause the diluted capture solution
to look paler than the undiluted capture solution.
N OTE 2—Highly colored or hazy samples may color the capture
solution This will not affect the RLU reading.
11.1.10 Ensure that the luminometer is powered on and has
successfully completed the self-check and is ready for analysis
11.1.11 After the 5 min standing as prescribed in11.1.9,
11.1.11.1 Obtain the large pipette from the fuel test kit,
11.1.11.2 Using a clean implement (for example, scissors or
knife), cut the plastic protective sleeve open at the bulb-end
and remove the pipette Do not touch the tip and lower stem of
the pipette by hand or against any surfaces
11.1.12 Coalesce the capture solution and any water phase
present into a single drop or phase and use the large pipette to
retrieve and transfer a sample aliquant to the fuel test pen
reservoir for testing
11.1.13 The level of capture solution sample must at least
reach up to the bottom of the bowl shape on the sampling tube
If excess fuel phase (more than 1 mm visible above blue phase)
enters the sampling tube, use the same pipette to remove free
fuel prior to testing
11.1.14 Close the lid on the sampling tube reservoir and
separate the sampling pen from the sampling tube to expose the
sterile white sampling stick
11.1.14.1 Do not touch the sampling stick by hand or
against any surface
11.1.15 Open the lid of the fuel test pen to access the
reservoir and dip the sampling stick into the capture solution
until it touches the bottom of the tube
11.1.16 Holding the tube and reservoir vertically, carefully
remove the sampling stick from the reservoir without touching
the stick against the sides of the sampling tube
11.1.17 Keep the pen vertical and sampling stick pointing
downward Do not shake the pen The capture solution should
be thoroughly deposited between all the ridges on the lower
half of the sampling stick
N OTE 3—If the capture solution sample contained excess fuel, this will
show up as white or pale “patches” in the rings of blue liquid.
11.1.17.1 If fuel is present, dip the stick again and move up
and down several times in the capture solution before removing
the sampling stick again This will usually rinse off most of the
fuel phase and ensure that the capture solution is evident on all
of the ridges of the sample stick
11.1.18 With the white sample stick pointing down, hold the pen cuvette firmly in a fist and firmly press the tip of the sampling stick vertically against a hard, flat, level surface, until the sampling stick retracts completely into the pen cuvette (chamber)
11.1.19 Activate the pen by pressing and turning the white pen collar clock-wise until finger-tight
11.1.20 Hold the sample cuvette between the thumb and forefinger and shake it in an end-to-end motion for 10 s to 20 s
N OTE 4—Good shaking of the pen is crucial to obtain complete reconstitution and mixing of the freeze-dried reagents deposited in the pen cap Insufficient mixing can typically be diagnosed by the light signal increasing with time for up to several minutes after the initial measure-ment.
11.1.20.1 Remove gloves that may have been worn during steps that involve potential direct contact with sample This will prevent the risk of static discharge while inserting test-pen into luminometer
11.1.21 Place the pen in the cuvette holder of the luminom-eter and close the lid to initiate the reading
11.1.21.1 Measure the light signal immediately after activa-tion of the test pens Results are displayed and the instrument lid opens after approximately 15 s
11.1.22 Record RLU
11.1.23 Remove the pen and discard according to local regulations
11.1.24 Calculate and report the results as described in Section12
12 Calculation and Report
12.1 A report should contain all relevant information such
as time, date, and exact sampling site and sampling method (sample volume, position in tank, and so forth) as well as the result reported in measured RLU and calculated as applicable 12.2 The result is displayed in RLU The measured signal is directly proportional to the concentration of ATP in the capture solution (or fuel-associated water phase) tested
12.3 For Fuel-Only samples, results reported in RLU are associated with microbial activity in suspended water or microliter water droplets contained in the 500 mL sample The precision statement in13.1.3 Fuel-Only applies
12.4 For Fuel/Water mixture or Water-Only samples, results reported in RLU are associated with microbial activity present
in the 1 mL test specimen obtained from the bulk water layer
in the sample bottle The precision statements in 13.1.3
Fuel/Water Mixture or Water-Only will apply respectively 12.5 For aviation fuel samples, results reported in RLU are associated with the microorganisms present in the 1000 mL sample Results are reported in accordance with applicable administrative guideline(s) and no precision statement applies 12.6 If the test results are >99 999 RLU or sample matrix interferences are suspected, then dilute the sample as instructed
inAppendix X4and correct the results for dilution using the following equation No precision statement applies
Trang 6this can be set to 20.
12.7 To determine cellular ATP, follow Section 11 and
Appendix X3 to determine Total RLU and Extracellular ATP
respectively:
RLU Cellular 5 RLU Total 2 RLU Extracellular (2)
13 Precision and Bias 5
13.1 Precision—The following precision was determined in
accordance with PracticeD6300
13.1.1 Repeatability—The difference between repetitive
re-sults obtained by the same operator in a given laboratory
applying the same test method with the same apparatus under
constant operating conditions on identical test material within
short intervals of time would in the long run, in the normal and
correct operation of the test method, exceed the values
deter-mined using the equations in13.1.3 only in 1 case in 20
13.1.2 Intermediate Precision—Between Operator/
Apparatus Repeatability: The difference between two single
and independent results obtained by different operators
apply-ing the same test method in same laboratory usapply-ing different
Repeatability 5 0.6584 · X1.05 RLU
Between Operator/Apparatus Repeatability 5 0.7038 · X1.05 RLU
13.1.3.3 Fuel-Only:
Repeatability 5 0.763 · X RLU Between Operator/Apparatus Repeatability 5 0.763 · X RLU
where:
X = the average of the two results.
13.2 Reproducibility—Due to the instability of the
mea-surand of this test method over time, it is not possible to determine reproducibility of this test method using traditional interlaboratory studies
13.3 Bias—Since there is no accepted reference material
suitable for determining the bias of this test method, bias cannot be determined
14 Keywords
14.1 adenosine triphosphate; assay; ATP; ATP Biolumines-cence Assay; bacteria; biocontamination; biodeterioration; bio-mass; capture solution; cellular; extracellular; fuel; fungi; hydrophilic particles; luciferin; luciferase; luminometer; mi-crobe; microbiology; Relative Light Unit (RLU)
APPENDIXES
(Nonmandatory Information) X1 DETERMINATION OF FUEL PHASE ATP (FUEL/WATER SAMPLES WITH ≥10 mL FREE-WATER)
X1.1 To remove free-water from sample:
X1.1.1 Transfer sample to an appropriate volume, sterile
separatory funnel and drain bottom-water and invert-emulsion
into a clean, sterile vessel
X1.1.2 Alternatively, use a sterile pipette to draw
bottom-water and invert-emulsion into a clean, sterile vessel or sample
bottle
X1.2 To determine ATP in the water fraction proceed to
Appendix X2 X1.3 To determine the ATP in the fuel fraction, follow steps
11.1.1through11.1.23 X1.4 Calculate and report the results as described in Section
12
5 Supporting data have been filed at ASTM International Headquarters and may
be obtained by requesting Research Report RR:D02-1812 Contact ASTM Customer
Service at service@astm.org.
Trang 7X2 DETERMINATION OF WATER-PHASE ATP (FUEL/WATER SAMPLES WITH ≥10 mL FREE-WATER)
X2.1 Separate bottom-water and invert-emulsion from
sample in accordance withX1.1
X2.2 Complete steps11.1.1.1though11.1.8
X2.3 Then complete steps11.1.11.1through11.1.23 X2.4 Calculate and report the results as described in Section
12
X3 DETERMINATION OF EXTRACELLULAR ATP
X3.1 This test is performed when it is desired to
differen-tiate between total, extracellular and, cell-associated ATP
present in the sample
X3.2 If a sample is to be tested for extracellular ATP, it is
essential to complete the sampling and testing with the free
ATP pen before sampling and testing using the fuel test pen
For further information see the discussion under3.1.10
X3.3 Prepare the sample as described in Section11
X3.4 To test for extracellular ATP, remove the extracellular free pen from its protective sleeve
X3.5 Complete the steps in11.1.14to11.1.23using the free ATP pens
X3.6 Calculate and report the results as described in Section
12
X4 RESOLVING MATRIX INTERFERENCES IN WATER SAMPLES
X4.1 Prepare an 11-fold dilution by adding 100 µL of
bottom water or of a previously tested capture solution sample
directly to the capture solution in the reservoir of a new fuel
test pen (8.2.1.1)
X4.2 Close the lid and mix the solution by turning the
reservoir horizontal to vertical several
X4.3 Proceed with the steps in11.1.14to11.1.23
X4.4 If required, higher dilutions can be prepared by reducing the volume of sample transferred to the 1.0 mL capture solution in the reservoir, for example, 50 µL for a 21-fold dilution 10 µL for a 101-fold dilution
X4.5 Proceed with the steps in11.1.14 to11.1.23 X4.6 Calculate and report the results as described in Section
12
X5 STANDARD CURVE, METHOD
X5.1 Prepare stock solutions from ATP di-sodium salt using
ATP free water Suggested stock solutions: (10–2 M, 10–4 M,)
10–6 M
N OTE X5.1—Stock solutions can be stored at –20°C dispensed in
ATP-free micro-centrifuge tubes.
X5.2 Prepare dilution series of 10–6 M stock solution to
make up the following ATP solutions: 3 × 10–8M, 10–8M, 3 ×
10–9M, 10–9M, 3 × 10–10M, 10–10M, 3 × 10–11M, 10–11M,
0 M
X5.3 For a reduced number of tests, it is recommended to
test the following concentrations: 3 × 10–8 M, 10–8 M, 3 ×
10–10M, 0 M
X5.4 Dispense 5 × 1.0 mL aliquots of each ATP (Sodium Salt) solution into sterile, ATP free 1.5 mL micro-centrifuge tubes
N OTE X5.2—For repeatability calculations, a minimum of five repeats
of each concentration is recommended.
X5.5 For blank measurements and diluent use ATP free water (Pharmaceutical Grade: pyrogen free water)
X5.6 For capture solution background measurements use Merck blue capture solution from fuel test pens.2
X5.7 Test each vial once by sampling with a fuel test pen.2
Trang 8SUMMARY OF CHANGES
Subcommittee D02.14 has identified the location of selected changes to this standard since the last issue (D7463 – 15) that may impact the use of this standard (Approved June 1, 2016.)
(1) Reinstate the legend to performEq 1
Subcommittee D02.14 has identified the location of selected changes to this standard since the last issue (D7463 – 14a) that may impact the use of this standard (Approved Dec 1, 2015.)
(1) Revised Sections2,7,9,11,12,14, andTable X6.1
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